Varicella-zoster virus (VZV) causes chickenpox and herpes zoster. We have developed a system to generate recombinant VZV by transfecting tissue culture cells with four overlapping VZV cosmid DNAs. The recombinant VZV will be useful to study the role of individual VZV genes in infection. Mutations have been engineered into cosmids that yield VZV mutants that are unable to express the viral thymidylate synthetase, glycoprotein V ribonucleotide reductase, and ORF10 genes. Virus deleted for the ribonucleotide reductase gene grows at a slower rate than wild-type virus in vitro and is more sensitive to acyclovir than parental virus. While the HSV-1 homolog of ORF10, VP16, is essential for replication of HSV-1 in cell culture, deletion of ORF10 from VZV does not result in impaired growth in vitro. Selected VZV mutants will be used to inoculate guinea pigs to determine if the viruses have lost the ability to establish latency in the central nervous system. The E. coli beta-galactosidase gene, has been inserted into the VZV genome, and the resultant virus produces plaques that stain blue with X gal. Guinea pigs are being inoculated with this virus to determine the types of cells that are able to support viral replication and establishment of latency. The herpes simplex virus (HSV) glycoprotein D (gD) gene, encoding a major neutralizing antigen, has been inserted into VZV and the resulting virus expresses high levels of HSV gD on the surface of infected cells. Inoculation of guinea pigs with this VZV mutant resulted in development of neutralizing antibodies to HSV-1. When the animals were challenged with live herpes simplex virus they showed reduced severity of herpes simplex infection compared with control animals. Two combinatorial libraries of E. coli expressing human Fab fragments that recognize VZV have been generated and screened for neutralizing activity against VZV.